201116828 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種化學式（I)化合物，以及利用該 化學式（I)化合物以偵測癌症細胞的方法。 【先前技術】 癌症是一種病徵在於其轉形細胞不規則細胞增生的 疾病，其會入侵並毀壞正常身體組織。儘管目前有許多癌 症療法正穩定發展中，但癌症仍為目前主要死因。目前研 究顯示’癌症的早期診斷可增進癌症療效，並因而減少了 癌症病患的死亡率。因此，找出一種有用的試劑，其可應 用於快速且正確的早期癌症診斷方法，是一件相當重要的 事。 【發明内容】 本發明係透過某些咔唑化合物與DNA接觸後，意 地發現，在癌症細胞中表現出的螢光強度較在正常細= 強，特別是在細胞核及粒線體中此情況更顯著。 因此，本發明係關於含有化學式（1)化合物 類化合物，其中前述化學式（G為：201116828 VI. Description of the Invention: [Technical Field] The present invention relates to a compound of the formula (I), and a method of using the compound of the formula (I) to detect cancer cells. [Prior Art] Cancer is a disease characterized by irregular cell proliferation of transformed cells, which invades and destroys normal body tissues. Although many cancer therapies are currently developing steadily, cancer remains the leading cause of death. Current research shows that early diagnosis of cancer can improve the efficacy of cancer and thus reduce the mortality rate of cancer patients. Therefore, it is important to find a useful reagent that can be applied to fast and correct early cancer diagnosis methods. SUMMARY OF THE INVENTION In the present invention, after contact with DNA by certain carbazole compounds, it is found that the fluorescence intensity exhibited in cancer cells is stronger than that in normal fines, especially in the nucleus and mitochondria. Significant. Accordingly, the present invention relates to a compound containing a compound of the formula (1) wherein the aforementioned chemical formula (G is:
201116828 其中，前述每一 A和B係獨立地為含有至少一個氮 原子之雜芳基（heteroaryl); 前述每一 X和Y係獨立地為CH或氮（N); 前述每一RrR6係獨立地為氫（H)、Ci_C8烷基、C2_C8 烯基、c2-c8炔基、c3-c8環烷基、C3_C8雜環烷基、芳基 (aryl)、雜芳基、羥基、Cl_C6烷氧基、芳氧基、雜芳氧基、 胺基 C〗_C6 烧胺基、一烧胺基（C!-C6 dialkylamino )、 芳胺基、二芳胺基、硫基或鹵素；前述1是Ci_Ci6烷基、 ^2-C16烯基、c2-C16炔基、C3-C8環烷基、c3-C8雜環烷基、 芳基、雜芳基、-C2H4_(ZC2H4)V-，其中前述/係為氧（〇)、 硫（S)/硒（Se)，且前述v係為卜2或3，前述R8係為 含有一氮原子之Cs-C8雜環烷基、經一胺基取代之C3_C8 雜，烷基、經一胺基取代之eta環烷基、經一胺基取代 之a芳基、含有一氮原子之雜芳基或NRaRb，其中前述每一 Μ和Rb係獨立地為氫、Cl_C8烷基，或Ra和…與氮原子 共同連接至一 3至8環原子所組成之環；且前述每、一爪和 η係獨立地為1、2或3。 —前述分子式之化合物可具有一或多種下列特性：前述 母Α和Β係獨立地為含有一個或二個氮原子之雜芳基 (例如：吡啶基或嘧啶基）。A或B之例子包含，但不限於： $烷基取代之吡啶基（例如：丨_甲基_嘧啶鏽_4_基 Hmethyl-PyHdinium_4_yl))。前述每一爪及11係為卜前 =母一心-R6係為氫。前述每一 χ和γ係為CH。前述尺 „ c8-cI2院基、c8_Ci2稀基、c8_Ci2块基、或_咖: _出4) 3_。R8係為含有一氮原子之雜環烷基，其可帶正 J 例性之R8包含，但不限於：含有一氮原子：雜環烷 土（例如.基-派咬鑌小基）。名詞「炫基」係指直鏈 201116828 或支鏈烴基（hydrocarbon )。示例性之炫基包含，但不限於： 甲基、乙基、正丙基、異丙基、正丁基、異丁基及第三丁 基。名詞「烯基」係指直鏈或支鏈烴基，且其含有一或多 個雙鍵。示例性之烯基包含，但不限於：乙烯基、丙烯基、 2-丙烯基（allyl)及1，4-丁二烯基。名詞「炔基」係指直鏈 或支鏈烴基，且其含有一或多個参鍵。示例性之炔基包含， 但不限於：乙炔基、1-丙炔基、1-和2-丁炔基及1-甲基-2-丁炔基。名詞「烷氧基」係指「-0-烷基」殘基。示例性之 烷氧基包含，但不限於：曱氧基、乙氧基、正丙氧基 (n_propoxy )、異丙氧基（isopropoxy )、正 丁氧基 (n-butoxy)、異丁氧基（iso-butoxy)、第二丁氧基及第三 丁氧基。 名詞「環烧基」係指具由3至8個碳原子組成之飽和 煙環狀系統。示例性之環烷基包含，但不限於：環丙基、 環丁基、環戊基、環己基、環庚基和環戊基。名詞「雜環 院基」係指具有一或多個雜原子（例如：氧、氮、硫或硒） 之飽和環狀基元（moiety)。示例性之雜環烷基團包含，但 不限於：哌嗪基（piperazinyl)、吡咯啶基（pyrr〇lidinyl)、 二噁烷基（dioxanyl)、嗎啉基（morpholinyl)及四氫呋喃 基（tetrahydrofuranyl)。 名詞「芳香基」係指一 6碳單環、丨〇碳雙環、丨4碳三 級之芳香環系統。示例性之芳香基圑包含，但不限於：苯 卞（phenyl)、秦基（naphthyl)、蔥基（anthracenyi)。名詞 芳氧基」係指「-〇-芳基」殘基。示例性的芳氧基包含， 值不限於：笨氧基（phen〇Xy)。㈣「雜*基」係指具有 或多個雜原子（例如：氧、氮、琉或石西）之5至8個環 原子的芳香單環、8_12個環原子的雙環或u_l4個環原子 201116828 的三環系統。示例性之雜芳基包含，但不限於：n比咬基 (pyridyl)、°夫σ南基（furyl)、味唾基（imidaz〇iyi)、苯并 咪唑基（benzimidazolyl )、嘧啶基（pyrimidinyl )、噻吩 (thienyl)、喹啉基（quinolinyl)、吲哚基（ind〇lyl)、噻唑 基（thiazolyl)。名詞「雜芳氧基」係指一 _〇_雜芳氧基 (-O-heteroary 1)殘基。示例性之雜芳烷基包含，但不限於： 4-。比。定氧基。 於本文中^51及之烧基、烯基、炔基、環烧基、雜環炫 基、芳香基、雜芳基、芳氧基、及雜芳氧基皆含有經取代 或未經取代的基元。示例性之取代基包含，但不限於：鹵 素、羥基、胺基、氰基、硝基、硫基、烷氧羰基、胺基、 烷磺醯基（alkanesulfonyl)、烷羰基（aikylcarbonyi)、脲基 (carbamido)、胺甲醯基（carbamoyl)、羧基（carb〇xy)、 硫脲基（thioureido )、氰硫基（thiocyanato )、續醯胺基 (sulfonamido)、烧基、稀基、炔基、烧氧基、芳基、雜芳 基及雜環烷基。環烷基及雜環烷基可與芳基或雜芳基結合。201116828 wherein each of the foregoing A and B is independently a heteroaryl containing at least one nitrogen atom; each of the foregoing X and Y systems is independently CH or nitrogen (N); each of the foregoing RrR6 is independently Is hydrogen (H), Ci_C8 alkyl, C2_C8 alkenyl, c2-c8 alkynyl, c3-c8 cycloalkyl, C3_C8 heterocycloalkyl, aryl, heteroaryl, hydroxy, Cl_C6 alkoxy, An aryloxy group, a heteroaryloxy group, an amine group C__C6 an amine group, an amine group (C!-C6 dialkylamino), an arylamine group, a diarylamino group, a thio group or a halogen; the aforementioned 1 is a Ci_Ci6 alkyl group , ^2-C16 alkenyl, c2-C16 alkynyl, C3-C8 cycloalkyl, c3-C8 heterocycloalkyl, aryl, heteroaryl, -C2H4_(ZC2H4)V-, wherein the aforementioned / is oxygen (〇), sulfur (S) / selenium (Se), and the aforementioned v is 2 or 3, and the above R8 is a Cs-C8 heterocycloalkyl group having a nitrogen atom, and a C3_C8 impurity substituted with an amine group. An alkyl group, an aryl group substituted with an amine group, an a aryl group substituted with an amine group, a heteroaryl group containing a nitrogen atom or NRaRb, wherein each of the above hydrazines and Rb is independently hydrogen, Cl_C8 alkane a base, or Ra and ... together with a nitrogen atom to a 3 to a ring composed of 8 ring atoms; and each of the above-mentioned one and one of the claws and η is independently 1, 2 or 3. - The compound of the above formula may have one or more of the following characteristics: The aforementioned parent and oxime are independently heteroaryl groups containing one or two nitrogen atoms (e.g., pyridyl or pyrimidinyl). Examples of A or B include, but are not limited to: an alkyl substituted pyridyl group (e.g., 丨_methyl_pyrimidine rust_4_yl Hmethyl-PyHdinium_4_yl)). Each of the above-mentioned claws and the 11th line is a pre-b = one-heart-R6 system is hydrogen. Each of the foregoing χ and γ systems is CH. The above-mentioned ruler c8-cI2 yard base, c8_Ci2 base group, c8_Ci2 block base, or _coffee: _out 4) 3_. R8 is a heterocyclic alkyl group containing a nitrogen atom, which can be positively represented by R8. However, it is not limited to: containing a nitrogen atom: heterocycloalkane (for example, a base group). The term "dacro" refers to a straight chain 201116828 or a branched hydrocarbon group. Exemplary bristles include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. The term "alkenyl" refers to a straight or branched chain hydrocarbon radical and which contains one or more double bonds. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, 2-allyl, and 1,4-butadienyl. The term "alkynyl" refers to a straight or branched chain hydrocarbon radical and which contains one or more reference bonds. Exemplary alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1- and 2-butynyl, and 1-methyl-2-butynyl. The term "alkoxy" refers to a "-0-alkyl" residue. Exemplary alkoxy groups include, but are not limited to, decyloxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy (iso-butoxy), second butoxy and tert-butoxy. The term "cycloalkyl" refers to a saturated smoke ring system consisting of 3 to 8 carbon atoms. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclopentyl. The term "heterocyclic" refers to a saturated cyclic moiety having one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, or selenium). Exemplary heterocycloalkyl groups include, but are not limited to, piperazinyl, pyrr〇lidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl. . The term "aromatic group" means a 6-carbon monocyclic, fluorene-bicyclic, 丨4 carbon tertiary aromatic ring system. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyi. The term "aryloxy" refers to a "-〇-aryl" residue. Exemplary aryloxy groups include, the value is not limited to: phenoxy (phen 〇 Xy). (d) "hetero* group" means an aromatic monocyclic ring of 5 to 8 ring atoms having one or more heteroatoms (for example, oxygen, nitrogen, helium or tarax), a bicyclic ring of 8 to 12 ring atoms or a ring of u_l4 rings 201116828 The three-ring system. Exemplary heteroaryl groups include, but are not limited to, n-pyridyl, furyl, imidaz〇iyi, benzimidazolyl, pyrimidinyl ), thiophene, quinolinyl, ind〇lyl, thiazolyl. The term "heteroaryloxy" means a residue of -O-heteroary 1 . Exemplary heteroarylalkyl groups include, but are not limited to: 4-. ratio. Alkoxy. As used herein, the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, aryloxy, and heteroaryloxy groups are substituted or unsubstituted. Primitive. Exemplary substituents include, but are not limited to, halogen, hydroxy, amine, cyano, nitro, thio, alkoxycarbonyl, amine, alkanesulfonyl, aikylcarbonyi, ureido (carbamido), carbamoyl, carb〇xy, thioureido, thiocyanato, sulfonamido, alkyl, dilute, alkynyl, Alkoxy, aryl, heteroaryl and heterocycloalkyl. The cycloalkyl and heterocycloalkyl groups can be bonded to an aryl or heteroaryl group.
201116828201116828
201116828 酸鹽、過氯酸鹽、六氟填酸鹽、項酸鹽、麟酸鹽、擰檬酸 鹽、甲基磺酸鹽（methanesulfonate )、三It乙酸鹽 (trifluoroacetate)或乙酸鹽。同樣地，鹽類亦可形成於一 陽離子及咔唑化合物的負電基團之間。適用之陽離子包 含：鈉離子、卸離子、鎮離子、#5離子及錢離子（例如： 四甲基銨離子）。 另一方面，本發明提供一種偵測培養而得或病人檢體 中的癌症細胞的方法。其方法包含：（1)將檢體中一些細 胞與前述化學式之化合物進行接觸，（2)偵測前述細胞所 發出之螢光，及（3)判斷檢體中是否含有癌症細胞。若前 述螢光的強度大於非癌症細胞所發出的螢光強度，則此檢 體含有癌症細胞。 本發明更多示例性態樣將由後文之圖式及說明詳述 之。本發明之其他特性、標的及優點顯見於說明書及專利 申請範圍中。 【實施方式】 我們進而設計一些咔唑化合物，在咔唑基元的9號位 置連結帶正電的含氮基團，此連結係透過一連結體（linker) 所組成。由於此系列分子與癌症細胞作用，多能進入細胞 核和粒線體，且與其内的DNA作用，因此所發出的螢光強 度較在一般正常細胞強許多。 據此，本文所描述的是以前述化學式I表示的咔唑化 合物，其皆含有與9號位置連結之帶正電的含氮基團（即 r8 )。這些化合物亦包含具有高達16個碳的連結體（即 R7)。前述連結體可單獨或與其他取代基共同存在，以調 整前述α卡t!坐化合物之親脂性/親水性。 若欲合成前述化合物，可利用合成化學轉型技術 201116828 (synthetic chemistry transformations)(包含保護基團方法 學 protecting group methodologies) ，iH -k〇 : R. Larock,201116828 Acid, perchlorate, hexafluoro-salt, sulphate, sulphate, sulphate, methanesulfonate, trifluoroacetate or acetate. Similarly, salts can also be formed between the cation and the negatively charged group of the carbazole compound. Suitable cations include: sodium ions, deionized ions, town ions, #5 ions, and money ions (eg, tetramethylammonium ions). In another aspect, the invention provides a method of detecting cancer cells in a cultured or patient sample. The method comprises the following steps: (1) contacting some cells in the sample with a compound of the above formula, (2) detecting the fluorescence emitted by the cells, and (3) determining whether the sample contains cancer cells. If the intensity of the aforementioned fluorescence is greater than the intensity of fluorescence emitted by non-cancer cells, the sample contains cancer cells. Further exemplary aspects of the present invention will be described in detail by the drawings and description below. Other characteristics, objects, and advantages of the invention are apparent from the description and the scope of the patent application. [Embodiment] We further designed some carbazole compounds, which are linked to a positively charged nitrogen-containing group at the 9th position of the carbazole moiety, and the bond is composed of a linker. Because this series of molecules interacts with cancer cells, they can enter the nucleus and mitochondria and interact with the DNA inside them, so the fluorescence intensity is much stronger than that of normal cells. Accordingly, what is described herein is an oxazole compound represented by the above formula I, which all contain a positively charged nitrogen-containing group (i.e., r8) attached to position 9. These compounds also contain a linker (i.e., R7) having up to 16 carbons. The aforementioned linker may be present alone or in combination with other substituents to adjust the lipophilicity/hydrophilicity of the aforementioned α-carboxyl compound. To synthesize the above compounds, synthetic chemical transformation techniques 201116828 (including protective group methodologies), iH -k〇 : R. Larock, can be used.
Comprehensive Organic Transformations, VHC Publishers (1989 ) ； T. W. Greene and P. G. M Wuts, Protective Groups IN organic Synthesis, 3rd Ed., John Wiley and Sons ( 1999 i L. Fieser And M. Fieser, Fieser's Reagents for Organic Synthesis, John Wiley and Sons ( 1994) ； and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis^ John Wiley and Sons ( 1995)及其再版之著作。 下列流程圖一表示前述化合物1-7之示例性合成方 法。 流程圖一：Comprehensive Organic Transformations, VHC Publishers (1989); TW Greene and PG M Wuts, Protective Groups IN organic Synthesis, 3rd Ed., John Wiley and Sons (1999 i L. Fieser And M. Fieser, Fieser's Reagents for Organic Synthesis, John Wiley And Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis^ John Wiley and Sons (1995) and reprints thereof. The following Scheme 1 shows an exemplary synthesis of the aforementioned compounds 1-7. Flow chart 1:
流程圖一顯示一種合成3,6,9-三取代吟σ坐化合物的路 徑，其中3,6-二溴咔唑為反應起始物。可藉由標準取代反 應引入在9號位置上的取代基，而3和6號位置之修飾可 經由眾所周知之偶合反應（例如Heck或Stille反應）來達 201116828 成。可用含有氮原 之化合物1-7。 、土7°之N-烷化或質子化來形成所欲 中。 的詳細合成過程將分別詳述於實施例1-7 何其他適、高效能液相層析分離、結晶或任 物。 次叮進一步純化之前述合成出的咔唑化合 多個不Ά:含二雙鍵、’且可能進-步含有-或 αο τ因此它們可能為消旋物和消旋混合物、 举-鏡像異構物、獨立的非鏡像異構物、非鏡像異構混合 物，和順-或反-或Ε-或Ζ-雙鍵異構物。所有的此類異構物 形式已被納入考量。 本發明亦關於一種偵測檢體中癌症細胞的方法，期透 過將前述檢體中的細胞與前述任一咔唑化合物接觸，並在 細胞經激發光照射而發出螢光時進行偵測。若前述細胞所 發出之螢光強度高於非癌症細胞的螢光強度，則判定檢體 中含有癌症細胞。此方法具有高度靈敏性而可偵測單一癌 症細胞。 以適當的體外測試初步衡量本發明味嗤化合物在區 分癌症細胞和正常細胞的效果（參實施例9 )。 前述流程圖即可確切的達到本發明而不再以其他文 字贅述之。下列實施例僅為示例性表示，並非用以限制任 何其他未揭露之流程圖。本文中所有引用的出版物皆以其 全文作為參考文獻。 實施例1:化合物1的製備 在氮氣環境下，將氫化鈉（0.3 g, 12.5 mmol，Aldrich) 201116828 加至含有3,6-二>臭-911-11卡°坐化合物（2呂，6.1111111〇1，八1(11*丨(；11) 的DMF ( 10 ml)。將前述混合物攪拌5分鐘，再加入1，4-二溴丁烧（3.9 g，18 mmol, Aldrich)，將混合物回流12小 時。將利用快速層析分離（正己烷：乙酸乙酯為2 : 1 )得 到3, 6-二溴-9- ( 1-漠丁基）味嗤化合物（產率65% )。使 所得化合物（1.84g，4 mmol )和 °辰咬（0.68g，8mmol，Aldrich) 在乙醇（30ml)中的溶液於微量破化鈉存在的環境下回流 24小時。反應之產物以管柱層析（正己院/乙酸乙酯1:2) 純化之，並將之加至一含有乙酸Is ( II)( palladium ( II) acetate，0.005g，0.02mmol，Aldrich )和三-鄰-甲苯膦 (tri-o-tolyphosphine) (0.04g，0.13mmol，Aldrich)的高壓量 瓶中；接著添加三乙胺（triethylamine，5ml )，乙腈 (acetonitrile，10ml)及 4-乙稀吼咬（4-vinylpyridine，0.53g， 5mmol，Aldrich)後，灌入氮氣使之冒泡（bubbled)持續5 分鐘，將瓶口密封；反應在約105°C下持續48小時；收集 沉澱物，以水和二氣甲烷（CH2C12)萃取二次，以無水硫 酸鎂乾燥之，接著將之溶於四氫呋喃（THF)中，過濾之， 並將產物以丙_再結晶。將含有前述產物（〇.5g, 1 mmol) 的DMF溶液（10ml)以過量碘甲烷（CH3I)回流6小時， 將所得產物以曱醇再結晶二次（總產率為35%，30(TC )。]H NMR (400 MHz, DMSO-d6) 6:8.83 ( d, J =Hz, 4H ) , 8.62 (s, 2H) , 8.23 (d, J= 12.4Hz, 2H) , 8.20 ( d，J = 4.4Hz,Scheme 1 shows a route for the synthesis of a 3,6,9-trisubstituted ruthenium sigma compound in which 3,6-dibromocarbazole is the starting material for the reaction. Substituents at position 9 can be introduced by standard substitution reactions, and modifications at positions 3 and 6 can be made by well-known coupling reactions (e.g., Heck or Stille reaction) to reach 201116828. Compounds 1-7 containing a nitrogen source can be used. N-alkylation or protonation of 7° soil to form the desired. The detailed synthesis process will be detailed in Example 1-7, respectively, for other suitable, high performance liquid chromatography separations, crystallization or any. Further purification of the above-mentioned synthesized carbazole compound multiple oximes: containing two double bonds, 'and possibly further-containing - or αο τ, thus they may be racemates and racemic mixtures, lift-mirror isomerism , independent non-image isomers, non-imagewise isomeric mixtures, and cis- or trans- or Ε- or Ζ-double bond isomers. All such isomeric forms have been taken into account. The present invention also relates to a method for detecting cancer cells in a sample by contacting the cells in the sample with any of the aforementioned carbazole compounds and detecting when the cells are irradiated with excitation light to emit fluorescence. If the fluorescence intensity of the cells is higher than the fluorescence intensity of the non-cancer cells, it is determined that the specimen contains cancer cells. This method is highly sensitive and can detect single cancer cells. The effect of the miso compounds of the present invention on the differentiation of cancer cells and normal cells was initially measured by appropriate in vitro tests (see Example 9). The foregoing flow chart can be used to achieve the present invention without further recitation. The following examples are merely illustrative and are not intended to limit any other undisclosed flow charts. All publications cited herein are incorporated by reference in their entirety. Example 1: Preparation of Compound 1 Under a nitrogen atmosphere, sodium hydride (0.3 g, 12.5 mmol, Aldrich) 201116828 was added to a compound containing 3,6-di> odor-911-11 card (2 Lu, 6.1111111). 〇1, 八1 (11*丨(;11) in DMF (10 ml). The mixture was stirred for 5 minutes, then 1,4-dibromobutane (3.9 g, 18 mmol, Aldrich) was added and the mixture was refluxed. 12 hours. A 3,6-dibromo-9-(1-dibutylbutyl) miso compound (yield 65%) was obtained by flash chromatography (n-hexane: ethyl acetate: 2:1). A solution of the compound (1.84 g, 4 mmol) and EtOAc (0.68 g, 8 mmol, Aldrich) in ethanol (30 ml) was refluxed for 24 hours in the presence of a portion of sodium sulphate. Zhengxieyuan/ethyl acetate 1:2) purified and added to one containing acetic acid Is (II) (palladium (II) acetate, 0.005g, 0.02mmol, Aldrich) and tri-o-tolylphosphine (tri- O-tolyphosphine) (0.04g, 0.13mmol, Aldrich) in a high-pressure vial; followed by the addition of triethylamine (5ml), acetonitrile (10ml) and 4-ethyl benzoate (4-vinylp) After yridine, 0.53 g, 5 mmol, Aldrich), nitrogen was bubbled in for 5 minutes to seal the mouth of the bottle; the reaction was continued at about 105 ° C for 48 hours; the precipitate was collected with water and two gases. The methane (CH2C12) was extracted twice, dried over anhydrous magnesium sulfate, then dissolved in tetrahydrofuran (THF), filtered, and the product was recrystallized from hexanes. The DMF solution (10 ml) was refluxed with excess methyl iodide (CH3I) for 6 hours, and the obtained product was recrystallized twice from decyl alcohol (total yield: 35%, 30 (TC).] H NMR (400 MHz, DMSO-d6) 6:8.83 ( d, J = Hz, 4H ) , 8.62 (s, 2H) , 8.23 (d, J = 12.4Hz, 2H) , 8.20 ( d, J = 4.4Hz,
4H) , 7.96 (d, J = 8.4Hz, 2H) ,7.82 ( d, J = 8.4Hz, 2H), 7.57 (d, J= 15.6Hz, 2H) ,4.52 (t, 2H) , 4.25 ( s, 6H), 2.94 (s, 3H) , 2.25 ( t, 4H) , 2.21 ( t, 2H) , 1.84 ( m, 4H )，1.53 ( m，4H )，1.48 ( m, 2H ) . EA (C38H45I3N4.0.5H2O) : calc (obs%) C: 48.17 (48.03), 12 201116828 Η: 4.89 (4.91 ) , Ν: 5.91 ( 5.86) ° 實施例2 :化合物2的製備 化合物2的製備方式與前述實施例1相似。 整體產率：28 %，融點 > 300°C。4 NMR ( 400 MHz, DMSO-d6) 6 ： 8.80 ( d, J=6Hz, 4H) , 8.62 ( s, 2H) , 8.23 (d，J=16Hz，2H )，8.20 ( d，J=4.4Hz，4H )，7.93 ( d, J=8.8Hz， 2H) , 7.80 ( d, J=8.4Hz, 2H) , 7.53 ( d, J=16Hz, 2H) , 4.52 (t, 2H) , 4.25 (s, 6H) , 3.26 (m, 6H) , 1.89 ( m, 2H) , 1.73 (m，6H )，1.51 ( m, 2H ) , 1.34 ( m, 2H ) . EA (C39H47I3N4.0.5H2O) : calc(〇bs%) C :48.72 (48.59)， H ： 5.03 ( 5.02) , N ： 5.83 ( 5.75 ). 實施例3 :化合物3的製備 化合物3的製備方式與前述實施例1相似。 整體產率：31%，融點 > 300 °C。NMR ( 400 MHz， DMSO-d6) δ: 8.82 ( d，J = 6‘4Ηζ, 4Η )，8.63 ( s, 2H), 8.20 (d, J = 6.4Hz, 4H), 8.19 ( d, J = 16Hz, 2H), 7.92 ( d, J = 8.4Hz, 2H), 7.77 ( d, J = 8.4Hz, 2H), 7.55 (d, J=16Hz, 2H) 4.48 (t, 2H),4.24 (s, 6H),3.25 (m, 6H),2.94 , (s,3H),1.80 (m,2H),1.73 (m,4H),1.59 (m,2H),4H), 7.96 (d, J = 8.4Hz, 2H), 7.82 (d, J = 8.4Hz, 2H), 7.57 (d, J = 15.6Hz, 2H), 4.52 (t, 2H), 4.25 ( s, 6H), 2.94 (s, 3H) , 2.25 ( t, 4H) , 2.21 ( t, 2H) , 1.84 ( m, 4H ), 1.53 ( m, 4H ), 1.48 ( m, 2H ) . EA (C38H45I3N4.0.5 H2O) : calc (obs%) C: 48.17 (48.03), 12 201116828 Η: 4.89 (4.91 ) , Ν: 5.91 ( 5.86) ° Example 2: Preparation of Compound 2 Compound 2 was prepared in a similar manner to Example 1 above. . Overall yield: 28%, melting point > 300 °C. 4 NMR ( 400 MHz, DMSO-d6) 6 : 8.80 ( d, J = 6 Hz, 4H) , 8.62 ( s, 2H) , 8.23 (d, J = 16 Hz, 2H ), 8.20 (d, J = 4.4 Hz, 4H ), 7.93 ( d, J=8.8Hz, 2H) , 7.80 ( d, J=8.4Hz, 2H) , 7.53 ( d, J=16Hz, 2H) , 4.52 (t, 2H) , 4.25 (s, 6H ) , 3.26 (m, 6H) , 1.89 ( m, 2H) , 1.73 (m, 6H ), 1.51 ( m, 2H ) , 1.34 ( m, 2H ) . EA (C39H47I3N4.0.5H2O) : calc(〇bs% C: 48.72 (48.59), H: 5.03 (5.02), N: 5.83 ( 5.75 ). Example 3: Preparation of Compound 3 Compound 3 was prepared in a similar manner to Example 1 above. Overall yield: 31%, melting point > 300 °C. NMR ( 400 MHz, DMSO-d6) δ: 8.82 (d, J = 6'4 Ηζ, 4 Η ), 8.63 ( s, 2H), 8.20 (d, J = 6.4 Hz, 4H), 8.19 ( d, J = 16 Hz , 2H), 7.92 ( d, J = 8.4Hz, 2H), 7.77 ( d, J = 8.4Hz, 2H), 7.55 (d, J=16Hz, 2H) 4.48 (t, 2H), 4.24 (s, 6H) ), 3.25 (m, 6H), 2.94, (s, 3H), 1.80 (m, 2H), 1.73 (m, 4H), 1.59 (m, 2H),
1.50 (m, 2H) , 1.30 ( m, 4H) , 1.24 (m, 4H) . EA (C42H53I3N4.H20): caic (〇bs%) C: 49.82 (49.73 ), H ： 5.47 ( 5.43) , N ： 5.53 ( 5.45). 實施例4:化合物4的製備 化合物4的製備方式與前述實施例1相似。 13 201116828 整體產率：30%，融點 > 300 °C。^ NMR ( 400 MHz， DMSO-d6) δ： 8.81 ( d, J = 6.0Hz, 4H) , 8.67 ( s, 2H), 8.22 (d，J = 6.4Hz，4H)，8.20 (d，J= 16Hz，2H)，7.92 (d， J = 8.4Hz, 2H), 7.76 ( d, J = 8.8Hz, 2H), 7.58 ( d, J = 16Hz, 2H) , 4.47 (t, 2H) ,4.25 (s, 6H) ,3.28 (m, 6H) ,2.95 (s,3H),1.75 (m,4H),1.59 (m,4H),1.21 (m, 12H). EA (C43H55I3N4.1.5H20) : calc (obs %) C： 49.87 (49.79)，H: 5.64 ( 5.62)，N: 5.41 (5.35)。 實施例5 :化合物5的製備 化合物5的製備方式與前述實施例1相似。 整體產率：31%，融點 > 300 °C。咕 NMR ( 400 MHz, DMSO-d6) δ： 8.81 (d,J = 6.8Hz, 4H) ,8.65 ( s, 2H), 8.24 (d, J= 16Hz, 2H), 8.20 ( d, J = 7.2Hz, 4H ) , 7.92 ( d, J = 8.8Hz, 2H), 7.76 ( d, J = 8.8Hz, 2H), 7.56 (d, J=16Hz, 2H),4.45 (t,2H),4.29 (s,6H),3.25 (m,6H),2.95 (s, 3H), 1.75 (m, 6H), 1.61 ( m, 2H), 1.525( m, 2H),1.202 (m，16H) · EA (C46H61I3N4.H20) : calc (obs%) C: 51.70 (51.61)，H: 5.94 (5.91)，N: 5,24 (5.17)。 實施例6 :化合物6的製備 化合物6的製備方式與前述實施例1相似。以3,6,9· 二氧 Η 烧]，11 二漠化物（3,6,9-trioxaundecane-l，l 1-dibromide) > Soma et al., J. Phys. Chem. B, 1998, 102, 6152-6160 而製備。 整體產率：24%，融點 >3〇〇°c。nMR ( 400 MHz， DMSO-d6) δ： 8.80 ( d, J = 6Hz, 4H ) , 8.68 (s, 2H), 201116828 8.23 (d, J= 16Hz, 2H), 8.20 ( d, J = 7.2Hz, 4H), 7.90 ( d, J = 8.8Hz, 2H), 7.76 ( d, J = 8.4Hz, 2H), 7.59 (d, J=16Hz, 2H) ,4.64 (t, 2H) , 4.24 ( s, 6H) , 3.82 ( t, 2H) , 3.71 (t, 2H) , 3.47 (m，4H) , 3.38 (m，10H)，2.97 (s，3H)， 1.67 (m，4H),1.43 (m，2H).EA (C42H53I3N403.2H20): calc (obs%) C： 46.77 (46.68) ,H： 5.33 (5.31), N: 5.19 (5.12)。 實施例7 :化合物7的製備 化合物6的製備方式與前述實施例1相似。 整體產率：27%，融點 > 300 °C。NMR ( 400 MHz， DMSO-d6) δ: 8.94 ( s，2Η )，8.87 ( d，J = 6.4Hz，2Η ), 8.56 (d, J = 8.4Hz, 2H),8.46 ( t, J = 8Hz, 2H ) , 8.16 ( d, J = 16Hz, 2H) , 8.03 ( d, J = 8.8Hz, 2H) , 7.87 (t，2H)， 7.86 ( d, J = 8.4Hz, 2H ) , 7.67 ( d, J = 16Hz, 2H ) , 4.55 ( t, 2H)，4.45 (s，6H), 3.37 (m,6H)，2.99 (s，3H), 1.851.50 (m, 2H) , 1.30 ( m, 4H) , 1.24 (m, 4H) . EA (C42H53I3N4.H20): caic (〇bs%) C: 49.82 (49.73 ), H : 5.47 ( 5.43) , N : 5.53 ( 5.45). Example 4: Preparation of Compound 4 Compound 4 was prepared in a similar manner to Example 1 above. 13 201116828 Overall yield: 30%, melting point > 300 °C. ^ NMR ( 400 MHz, DMSO-d6) δ: 8.81 (d, J = 6.0 Hz, 4H), 8.67 (s, 2H), 8.22 (d, J = 6.4 Hz, 4H), 8.20 (d, J = 16 Hz , 2H), 7.92 (d, J = 8.4Hz, 2H), 7.76 ( d, J = 8.8Hz, 2H), 7.58 ( d, J = 16Hz, 2H) , 4.47 (t, 2H) , 4.25 (s, 6H) , 3.28 (m, 6H), 2.95 (s, 3H), 1.75 (m, 4H), 1.59 (m, 4H), 1.21 (m, 12H). EA (C43H55I3N4.1.5H20) : calc (obs % C: 49.87 (49.79), H: 5.64 ( 5.62), N: 5.41 (5.35). Example 5: Preparation of Compound 5 Compound 5 was prepared in a manner similar to that of Example 1 above. Overall yield: 31%, melting point > 300 °C.咕NMR (400 MHz, DMSO-d6) δ: 8.81 (d, J = 6.8 Hz, 4H), 8.65 (s, 2H), 8.24 (d, J = 16 Hz, 2H), 8.20 (d, J = 7.2 Hz , 4H ) , 7.92 ( d, J = 8.8Hz, 2H), 7.76 ( d, J = 8.8Hz, 2H), 7.56 (d, J=16Hz, 2H), 4.45 (t, 2H), 4.29 (s, 6H), 3.25 (m, 6H), 2.95 (s, 3H), 1.75 (m, 6H), 1.61 ( m, 2H), 1.525 ( m, 2H), 1.202 (m, 16H) · EA (C46H61I3N4.H20 ) : calc (obs%) C: 51.70 (51.61), H: 5.94 (5.91), N: 5, 24 (5.17). Example 6: Preparation of Compound 6 Compound 6 was prepared in a manner similar to that of Example 1 described above. 3,6,9· Dioxins], 11 Dioxin (3,6,9-trioxaundecane-l,l 1-dibromide) > Soma et al., J. Phys. Chem. B, 1998, Prepared by 102, 6152-6160. Overall yield: 24%, melting point > 3 〇〇 °c. nMR (400 MHz, DMSO-d6) δ: 8.80 (d, J = 6 Hz, 4H), 8.68 (s, 2H), 201116828 8.23 (d, J = 16 Hz, 2H), 8.20 (d, J = 7.2 Hz, 4H), 7.90 ( d, J = 8.8Hz, 2H), 7.76 ( d, J = 8.4Hz, 2H), 7.59 (d, J=16Hz, 2H) , 4.64 (t, 2H) , 4.24 ( s, 6H ), 3.82 (t, 2H), 3.71 (t, 2H), 3.47 (m, 4H), 3.38 (m, 10H), 2.97 (s, 3H), 1.67 (m, 4H), 1.43 (m, 2H) .EA (C42H53I3N403.2H20): calc (obs%) C: 46.77 (46.68), H: 5.33 (5.31), N: 5.19 (5.12). Example 7: Preparation of Compound 7 Compound 6 was prepared in a similar manner to Example 1 above. Overall yield: 27%, melting point > 300 °C. NMR (400 MHz, DMSO-d6) δ: 8.94 ( s, 2 Η ), 8.87 ( d, J = 6.4 Hz, 2 Η ), 8.56 (d, J = 8.4 Hz, 2H), 8.46 (t, J = 8 Hz, 2H ) , 8.16 ( d, J = 16Hz, 2H) , 8.03 ( d, J = 8.8Hz, 2H) , 7.87 (t, 2H), 7.86 ( d, J = 8.4Hz, 2H ) , 7.67 ( d, J = 16Hz, 2H ) , 4.55 ( t, 2H), 4.45 (s, 6H), 3.37 (m, 6H), 2.99 (s, 3H), 1.85
(m, 4H ) , 1.77 ( m, 4H ) , 1.52 ( m, 2H ) . EA (C38H45I3N4.0.5H2〇 ): calc ( obs % ) C: 48.17 ( 48.09 )， H : 4.89 ( 4.86)，N : 5.91 (5.88)。 實施例8 :咔唑化合物和線狀雙股DNA之間的連結 以美國公開專利第2005/0249669號及美國核准專利 第6,979,738號之方法測試咔唑化合物3、4、6和7與線 狀雙股 DNA ( linear duplex, LD)的連結。 LD之序列為： 5’-GCGCAATTGCGC-3,（ SEQ ID NO : 1 )。 化合物3、4、6和7的吸收光譜顯示吸收光譜帶有紅 201116828 色位移’且其莫耳吸收係數降低。這些改變表示前述化合 物與LD連結。化合物3、4、6和7的螢光光譜顯示，當 與DNA產生交互作用時，其螢光增強至少一個級數。田 實施例9 :癌症細胞診斷 以流式細胞儀測試化合物1-7對於區分clk肺癌細 胞及MRC-5正常人類肺纖維母細胞分化的效果。在癌症細 胞中’化合物1-7的平均螢光強度意外地較在正常細胞中 強。化合物3在癌症細胞與正常細胞間表現最顯著的差異。 測 Kang et al” J. C/zz>z. C/?ew· 5W” 2005, 52, 1069-1072 所述之3,6-雙（1-甲基-4-乙稀》»比咬）吁0坐二峨（3,6-bis (l-methyl-4-vinylpyridinium ) carbazole diiodide, BMVC 和化合物3在CL 1-0肺癌細胞及MRC-7正常人類肺纖維母 細胞中的螢光強度，而作為培養時間之函數❶本測試顯示 化合物3在癌症細胞中的螢光強度較BMVC的螢光強度更 強約3倍，且兩者化合物在正常細胞中都只表現出微弱的 螢光。 不僅如此’本實施例亦使用如美國公開專利第 2007/0098233 號及 Kang et al·，如α/_，2007, 132, 745-749 所述之裝置測試化合物3在區分CL1-0肺癌細胞和MRC-5 正常人類肺纖維母細胞的效果。化合物3在癌症細胞的螢 光強度明顯地比在正常細胞中為強。 實施例10 :親脂性 化合物卜6的親脂性取決於如Engelmann et al.，/咐.《/. P/zarm.，2007, 329, 12-18所述之η-辛醇/水係數的對數。化 合物1-6的log Ρ值界於-1.7與-2.4之間，顯示這些化合物 201116828 具有不同的親脂性。 實施例11 :細胞内分佈的位置 將CLK〇肺癌細胞培養於12孔培養皿中的蓋玻片上 24小時’以進行細胞内分佈位置的研究。24小時後，將i 或 5 μΜ 的化合物丨_7、50 nM 的 Mitotracker red CMXRos (M-7512, lnvitr〇gen)加入細胞培養液中，培養30分鐘。 接著’將4〇 nM Hoechst 33342 ( Sigma)加入細胞培養 液中’培養10分鐘，再以PBS清洗二次。以國立台大醫 15元之共輛焦顯微鏡（confocal microscope )( Leica TCS SP5 ) 來呈現細胞影像。以MetaMorph offline 7.6軟體來測定螢 光化合物U7和Mitotracker red之間重疊的區域。結果顯 不具有較高親脂性的化合物不會出現在癌症細胞的細胞 核，而位於粒線體中，這兩個地方會被Mit〇tracker 色。 其他實施態樣 人，在兒明書中所揭露的所有特徵都可能與其他方法衾 二本:兒明書中所揭露的每—個特徵都可能選擇性的以才I 1古相等或相似目的賴所取代，因此，除非有特別說明 ====揭==非用， c各種之更動_而； 述料化合物結構相似之 Τ用以實缺本發明。因此其他實施態彳_蓋 201116828 明之申請專利範圍之内。 18(m, 4H ) , 1.77 ( m, 4H ) , 1.52 ( m, 2H ) . EA (C38H45I3N4.0.5H2〇): calc ( obs % ) C: 48.17 ( 48.09 ), H : 4.89 ( 4.86), N : 5.91 (5.88). Example 8: The carbazole compound 3, 4, 6 and 7 and the linear double were tested by the method of U.S. Patent No. 2005/0249669 and U.S. Patent No. 6,979,738. Link of stranded DNA (LD). The sequence of LD is: 5'-GCGCAATTGCGC-3, (SEQ ID NO: 1). The absorption spectra of Compounds 3, 4, 6 and 7 showed an absorption spectrum with red 201116828 color shift' and a decrease in the molar absorption coefficient. These changes indicate that the aforementioned compound is linked to the LD. The fluorescence spectra of compounds 3, 4, 6 and 7 show that when interacting with DNA, their fluorescence is enhanced by at least one order. Field Example 9: Cancer cell diagnosis The effects of Compound 1-7 on differentiation of clk lung cancer cells and MRC-5 normal human lung fibroblasts were tested by flow cytometry. In the cancer cells, the average fluorescence intensity of Compound 1-7 was unexpectedly stronger than in normal cells. Compound 3 showed the most significant difference between cancer cells and normal cells. Measured by Kang et al" J. C/zz>z. C/?ew·5W" 2005, 52, 1069-1072 3,6-bis(1-methyl-4-ethene) than bite The fluorescence intensity of 3,6-bis (l-methyl-4-vinylpyridinium) carbazole diiodide, BMVC and compound 3 in CL 1-0 lung cancer cells and MRC-7 normal human lung fibroblasts, As a function of culture time, this test showed that the fluorescence intensity of Compound 3 in cancer cells was about 3 times stronger than that of BMVC, and both compounds showed only weak fluorescence in normal cells. Thus, the present embodiment also uses a device as described in U.S. Patent Publication No. 2007/0098233 and Kang et al., as described in α/_, 2007, 132, 745-749 to test compound 3 in distinguishing CL1-0 lung cancer cells from MRC. -5 Effect of normal human lung fibroblasts. The fluorescence intensity of Compound 3 in cancer cells is significantly stronger than in normal cells. Example 10: Lipophilicity of lipophilic compound 6 depends on, for example, Engelmann et al. /咐. The logarithm of the η-octanol/water coefficient as described in /. P/zarm., 2007, 329, 12-18. The log Ρ value of compound 1-6 is between -1.7 and - Between 2.4, these compounds 201116828 were shown to have different lipophilicity. Example 11: Location of intracellular distribution CLK〇 lung cancer cells were cultured on coverslips in 12-well culture dishes for 24 hours to conduct intracellular localization studies. After 24 hours, add i or 5 μΜ of compound 丨7, 50 nM Mitotracker red CMXRos (M-7512, lnvitr〇gen) to the cell culture medium and incubate for 30 minutes. Then '4〇nM Hoechst 33342 ( Sigma) was added to the cell culture medium for 'cultivation for 10 minutes, and then washed twice with PBS. The cell image was presented with a 15% confocal microscope (Leica TCS SP5) from National Taiwan University. The MetaMorph offline 7.6 software was used. The area overlapping between the fluorescent compound U7 and Mitotracker red was determined. As a result, the compound having no higher lipophilicity does not appear in the nucleus of the cancer cell, but in the mitochondria, the two places are colored by the Mitttracker color. Other implementations, all the features revealed in the children's books may be related to other methods: every feature disclosed in the children's book may be Selectively replaced by the same or similar purpose, therefore, unless otherwise specified ======================================================== this invention. Therefore, other implementation states are within the scope of the patent application of 201116828. 18